PROJECT SUMMARY/ABSTRACT Understanding how ribosomes choose translation initiation sites on mRNAs is an ongoing challenge in biology and is of great importance for investigation of cell function. We test the hypothesis that sequences just downstream of the translation start codon of mRNAs base pair with a conserved sequence in 18S rRNA termed the 530 loop that is located in the ribosome entrance tunnel for mRNAs. We propose that this base pairing helps position the AUG start codon at the ribosome P site facilitating translation initiation. This hypothesis is based on the observation that the 530 loop has base-pair complementarity to a three-nucleotide periodicity found in protein open reading frames and this periodicity is significantly enhanced 10-20 nucleotides downstream of the start codon of yeast proteins detected by peptide MS/MS as well as TAP-tagged proteins exhibiting high expression in large-scale western analysis. Using CRISPER-Cas9 gene editing, we will change the mRNA sequences 10-20 nucleotides downstream of the start codon to sequences that are not expected to base pair with the 530 loop and we will test using western analysis whether translation is depressed as predicted by the model. We will also test sequences that change the reading frame registration of the base pairing to assess whether the precise positioning of the base-paired mRNA is important for translation initiation. Enhancement of the same three-nucleotide periodicity is also found 10-20 nucleotides after non-standard start codons that initiate translation upstream or downstream of the annotated start. We will test whether reducing potential for base pairing by these sequences to the 530 loop down-regulates initiation at these non-standard start sites. Improving our understanding of translation initiation will be important in studying gene expression particularly in contexts where altered expression leads to gene disfunction, such as in disease states.